Project Summary Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease and Frontotemporal dementia (FTD) is the second most common form of early-onset dementia. Interestingly, both of these devastating neurodegenerative diseases share a common genetic mutation in chromosome 9 open reading frame 72 (C9orf72). An expanded hexanucleotide repeat (GGGGCC HRE) in intron 1 of the C9orf72 gene is the most common genetic cause of familial and sporadic ALS (sALS) and FTD, along with Huntington's disease phenocopies. Until recently, very little was known about the underlying mechanisms by which this expanded repeat causes neurodegeneration until 5 independent labs published 3 papers including one from our own group simultaneously showing that dysfunction in Nucleocytoplasmic Transport (NCT) may be a fundamental pathway for C9orf72-ALS/FTD pathogenesis. NCT, the trafficking of protein and RNA between the nucleus and cytoplasm, is critical for signal transduction and is especially arduous for neurons due to their highly polarized biology. Efficient regulation of this process is mediated by the Nuclear Pore Complex (NPC), an extraordinary molecular machine that serves as the main gateway to the nucleus. In order for any cell to function properly, it is imperative that RNA and protein be efficiently and selectively exchanged between the nucleus and the cytoplasm. This critical task is achieved by the ~2000 NPCs that span the entire nuclear envelope. Each NPC consists of multiple copies of 30 different proteins called Nucleoporins (NUPs) that differ in anatomical location, function, domain, post-translational modification and residence time. Mutations in various NUPs result in tissue-specific diseases. Additionally, some of the longest-lived proteins in the mammalian brain are specific NUPs and may represent the ?weakest link? in the aging proteome. We hypothesize that products of the C9orf72 repeat expansion are likely to disrupt NCT at the NPC and that NPC dysfunction may be a common pathogenic mechanism underlying the majority of ALS, not just C9orf72- ALS/FTD. This project will use immunohistochemistry, immunofluorescence, super resolution microscopy, and proteomics to characterize and quantify abnormalities in the NPC in C9orf72 and sALS human tissue, iPS cells, and various transgenic mouse models with the hope of identifying specific NUPs that are selectively affected, 2) assess NPC functionality using a dextran exclusion assay, 3) assess the pathophysiological roles of NPC glycosylation, expanded GGGGCC HRE, and/or Ran Translation Products as potential mechanisms in C9-ALS/FTD NPC dysfunction, and 4) determine the protein composition of NPCs across different CNS cell types in health and ALS using a novel approach to selectively isolate, purify, and characterize NPCs from mouse brain.